Anti pras40

Cells were lysed using complete radioimmune precipitation assay (RIPA) buffer supplemented with complete ULTRA protease inhibitor cocktail tablets (Roche, Basel, Switzerland) and sodium orthovanadate. Anti-Akt2, anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), anti-Pras40, anti-phospho-Pras40 (Thr246), anti-Gsk3A, anti-phospho-Gsk3 (Ser21/9), anti-Tsc2, anti-phospho-Tsc2 (Thr1426), anti-mTOR, anti-phospho-mTOR (Ser2448), anti-p70S6k1, anti-phospho-p70S6k1 (Thr389), anti-p70S6K2, anti-4E-BP1, anti-4E-BP1 (Thr37/46) (Cell Signalling Technology, Danver, Massachusetts, USA) rabbit polyclonal antibodies were used in immunoblotting. Luciferase constructs (pLightSwitch_3′UTR) (Switchgear genomics, Carlsbad, CA, USA) containing the 3′ UTR region of Akt2, Pras40, Gsk3a, CSF1 and Itgb1 was individually transfected into HEK293T cells using pGL4.12 (luc2CP) as a normaliser. Luciferase activity was measured by using the dual luciferase assay (Promega).

Total proteins in PC12 cells were isolated using RIPA Lysis Buffer (Beyotime Biotechnology). The protein concentration was determined using a BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China) with a full-wavelength functional microplate reader (Infinite M200Pro, Tecan, Switzerland). Then equal concentration Proteins were separated using 12.5% SDS–PAGE and transferred to nitrocellulose membranes. After blocking in 10% nonfat dry milk for 1 h, phosphorylated proteins were blocked using bovine serum albumin (5% BSA, room temperature) for 2 h. The membranes were incubated overnight at 4°C with the following primary antibodies: anti-phospho-mTOR-(Ser2448) (ZRB1553), anti-m-TOR(SAB5700687), anti-LC3 (SAB5701328) (Sigma Aldrich, United States), anti-phospho-PRAS40-(Thr246) (Cell Signaling) and anti-PRAS40(Cell Signaling)antibodies. The membrane was then incubated with the corresponding secondary antibody for 1 h. After three washes with PBST, the membrane was visualized using an ECL Western blot detection kit (Merck, United States). The average optical density of the images was analyzed using ImageJ.

Tumor and normal adjacent tissues were crushed with a mortar under liquid nitrogen and suspended on ice in lysis buffer, and the BCA Assay was used (Bio-Rad Laboratories, California, USA) to determine protein concentrations.
Harvested cells (1 × 10⁶) were homogenized in 100 μl RIPA lysis buffer (Solarbio, Beijing, China) supplemented with 1 × protease inhibitor cocktail. Proteins were separated using SDS-PAGE and transferred onto polyvinylidene fluoride membranes (PVDF, Immobilon P; Millipore, Billerica, MA, USA). After blocking in 5% low-fat milk powder in phosphate-buffered saline/Tween-20 (PBST) for 60 min, primary antibodies were incubated with the membranes at 4°C overnight. The antibodies were as follows: anti-PTEN (138G6), anti-AKT (C67E7), anti-p-AKT (Ser473) (D9E), anti-Cleaved caspase 3 (D175), anti-PRAS40 (D23C7), anti-p62/SQSTML (5114S), anti-LC3 (D3U4C), anti-BECLIN-1 (D40C5), anti-Bcl-xl (2764), anti-Bax (2772), anti-p-PRAS40 (Thr246) (D4D2) (Cell Signaling Technology, Danvers, MA, USA), and anti-GAPDH (sc-47724; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). After washing, membranes were incubated with the appropriate secondary antibodies (Santa Cruz Biotechnology) at room temperature for 45 min. Immunocomplexes were visible using an enhanced chemiluminescence detection system. Western blot results were analyzed using ImageJ (NIH).